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www.biomaghreb.com
IVD
FT En 45 Date of updating : 10/ 2020 Version B
IN VITRO DIAGNOSTIC USE
Manufacturer Use by In Vitro Diagnostic Temperature
Limitation
Catalogue number See insert Keep away
from light
Sufcient
for < n > essais
Batch number
CLINICAL SIGNIFICANCE
Syphilis is a venereal disease caused by T. pallidum infection. T. pallidum transmission occurs by
direct contact with a productive lesion. The incubation period is about 20 days and the disease
progress trough 3 different stages with different symptomatology. The anti-T. pallidum antibodies
appears in the first stage and may persist in the 85-90% of treated patients after they have been
treated and cured
PRINCIPLE
Avian erythrocytes sensilized with treponema palladium antigen will agglutinate in the presence of
serum anti treopnema antibodies to give characteristic patterns micro titration plate. No specific
reactions are defined by erythrocytes control (without sensitization).
REAGENT COMPOSITION (100 Tests)
Test Cells Stabilized avian erythocyte suspension sensitized
with T. pallidum antigen 1 vial of 7,5 ml
Control cells Stabilized avian cells suspension not
sensitized 1 vial of 7,5 ml
Diluant Buffer 1 vial of 20 ml
Positive control serum Pre-diluted at 1/20 1 vial of 1 ml
Negative control serum Pre-diluted at 1/20 1 vial of 1 ml
V.U. Well microtitration plates 3 plates (100 Tests)
*Serum :
Fresh serum, the serum can be stored at 2 - 8°C for 5 days, for longer periods, the serums should
be frozen (-20° C).
*Plasma :
EDTA plasma samples can be used for screening in blood banks.
SAFETY CAUTIONS
Biomaghreb reagents are intended for use by qualified personnel for in vitro use (do not pipette by
mouth).
• Refer to the current SDS available on request or at www.biomaghreb.com;
• Verify the integrity of the reagents before use; and
• Disposal of waste: comply with the legislation in force.
For safety reasons, treat any specimen or reagent of biological origin as potentially infectious.
Respect the legislation in force.
PRESERVATION AND STABILITY
At 2-8°C until the expiry date indicated on the label.
PROCEDURE
QUALITATIVE TEST :
• Well n° 1: add 10 µl of test serum to 190 µl of buffer to obtain a 1:20 dilution.
• Mix thoroughly: dispense 25 µl of this dilution into wells 2 and 3.
• Well n° 2 : add 75 µl of control cells
• Well n° 3: add 75 µl of test cells.
• Mix, cover the plate, wait 45 minutes at room temperature, away from any source of heat
and vibration.
Caution: Positive and negative control sera, which must be introduced in each series, are
pre-diluted to 1/20: make only cups 2 and 3.
QUANTITATIVE TEST
Well A B C D E F G H
Buffer 25 25 25 25 25 25 25 25
1/20 serum from cup
1 (μl)
25 25 25 25 25 25 25 25
Test Cells(μl)
75 75 75 75 75 75 75 75
Dilution 1/N 80 160 320 640 1280 2560 5120 10240
* Homogenize, cover the plate, and wait at least 45 minutes at room temperature away from all
sources of heat and vibration.
INTERPRETATION
- Read the results after 45 minutes.
- Results are stable for 24 hours.
- The images below correspond to reactions performed in U microplates.
4+ : Homogeneous veil of cells covering the entire well, sometimes with folded edges.
3+ : Homogeneous veil of cells partially covering the well bottom
2+: Veil of cells surrounded by a red circle of cells
1+ : Veil of cells surrounded by a thicker red circle of cells
± Cell button with a small central opening
-: Cell button with, or without, a very small central opening
Positive : from (4+) to (1+)
Doubtful : ±
Négative : -
For the interpretation of the results, it is imperative to compare them with the agglutinates of the
control sera.
Agglutination in well 2 of the screening test indicates the presence of non-specific agglutination.
In this case treat the serum as follows:
For 100 µl of serum, add 400 µl of control cells, shake, and wait 1 hour at laboratory temperature,
centrifuge for 5 min at 1000 rpm; repeat the test with the supernatant, bearing in mind that the serum
is diluted 1/5 by this treatment (redilute to 1/4 to obtain the equivalent of well 1).
The negative control must give a negative result.
The positive control should give a positive result in the qualitative test and react positively up to the
labelled titre ± double dilution in the quantitative test.
QUALITATIVE TEST :
A positive result indicates the presence of T pallidum antibodies resulting from past or present in-
fection.
A negative result indicates the absence of anti-T pallidum antibodies (see method limitations). A
questionable result in the qualitative test may correspond to low antibody levels in early stages of
syphilis or residual antibody levels in treated syphilis. In this case, an additional specimen may be
tested to demonstrate a possible increase in antibody titer.
QUANTITATIVE TEST :
The approximate titre will correspond to the last dilution giving a reaction.
LIMITS
* Although serum is the standard specimen for all syphilis tests, EDTA plasma samples can be used
for screening in blood banks. However, some reactions may be false positive. In this case, serum
should be re-tested on all initially positive or questionable tests.
* Specific antibodies can persist for a long time, even after successful treatment of the disease. In or-
der to estimate the response to treatment, the use of a reagin re-test (RPR re-test) is recommended
* TPHA may cross-react with other forms of treponemal infections and give false positive reactions
with specimens from patients with infectious mononucleosis, leprosy, drug addiction or autoim-
mune diseases
Occasionally, in some cases of early primary syphilis, specific antibodies may not be detected by the
TPHA technique.
TPHA
Haemagglutination Test for the
Serodiagnostic of Syphilis
